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Original Article Clinical significance of USP7 and EZH2 in predicting prognosis of laryngeal squamous cell carcinoma and their possible functional mechanism

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Original Article

Clinical significance of USP7 and EZH2 in predicting

prognosis of laryngeal squamous cell carcinoma

and their possible functional mechanism

Ming-Jie Zhang, De-Shang Chen, Hui Li, Wei-Wei Liu, Guo-Ying Han, Yue-Feng Han

Department of Otorhinolaryngology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui, China

Received February 4, 2019; Accepted February 26, 2019; Epub June 1, 2019; Published June 15, 2019

Abstract: Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most prevalent cancer types in the world. The ubiquitin specific protease 7 (USP7), a kind of deubiquitylating enzyme, has been reported to play multifaceted roles in different tumor types. EZH2 has been found to be highly expressed in various malignantcells and high expression of EZH2 is closely related to tumor growth infiltration, lymph node involvement, clinical stage, and poor prognosis. The aim of this study was to investigate the expression and function of USP7 and EZH2 in LSCC. Case presentation: Immunohistochemical staining and histochemical staining were performed to explore the expression of USP7 and EZH2 in both LSCC tissues and adjacent normal laryngeal tissues. Chi-square test, univari-ate analysis, and multivariunivari-ate analysis were conducted to statistically evaluunivari-ate the clinical significance of USP7 and EZH2. Conclusions: USP7and EZH2 affects LSCC evolution; USP7 and EZH2 were upregulated in LSCC tissues, which can serve as independent prognostic predictors, and potential therapeutic targets for LSCC.

Keywords: Laryngeal squamous cell carcinoma, USP7, EZH2, lymphatic invasion

Introduction

Laryngeal carcinoma is one of the most com-mon malignant tumors in otolaryngology, ac- counting for about 20% of head and neck tumors, 95% of which are mainly squamous cell carcinoma, mostly occurring in people over 40 years old [1]. Men’s rate is significantly higher than that of women, especially among middle-aged and elderly men middle-aged 60-70 years [2]. 177,442 new cases worldwide each year ac- count for 1% of cancer, and 94,713 deaths per year in 2018 [2]. Despite advances in diagnosis and treatment, the survival rate of patients with laryngeal cancer has not increased over the past 30 years, but has declined [3]. Patients with laryngeal cancer have various treatments, such as surgery, radiotherapy and chemothera-py, but the treatment effect and prognosis need to be improved [4].

Local recurrence, cervical lymph node metasta-sis, and poor differentiation are the main rea-sons for the treatment and prognosis of pa-

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cell-related receptors, key genes and regulato-ry molecules. Active components such as anti-bodies are targeted to cancer cells for the pur-pose of treating tumors. After targeted therapy, if only surgery is performed, the scope of tumor invasion gets reduced and the removed tissue will be less, so part of the laryngeal function can retained which brings better surgical prog-nosis to patients. In order to improve the thera-peutic effect of LSCC and preserve laryngeal function, exploring the relationship between tumor markers and clinicopathologic features of LSCC plays an important role in understand-ing the clinicopathologic features and treat-ment of LSCC.

USP7 (ubiquitin specific peptidase 7) is a deu-biquitinating enzyme that regulates many key proteins such as tumor suppressor genes, DNA repair proteins, immune responders, viral proteins, and epigenetic regulators [6]. USP7 abnormalities can cause tumor and viral dis-eases, therefore, it has become a potential therapeutic target [7]. USP7, also known as her-pes virus-associated ubiquitin-specific prote-ase (HAUSP), is reported to regulate DNA dam-age repair [8], DNA replication, cell mitosis [9], viral infection [10]. USP7 has also been report-ed to be involvreport-ed in tumor progression. The overexpression of USP7 has been observed in cancer, myeloma cells, neuroblastoma, breast cancer, non-small cell lung cancer, and ovarian cancer [11-15]. USP7 inhibitor can restrain pro-gression of colorectal tumor and non-small cell lung cancer [16, 17].

Enhancer of Zeste homolog 2 (EZH2), which is part of the core subunit of the polycomb repres-sive complex 2 (PRC2), catalyzes the transcrip-tion of its downstream target gene by catalytic methylation modification of histone H3K27 (H3K27me3) [18]. H3K27me3 always inhibits transcription, regulates gene activity at the chromosome level and it is a critical epigenetic event during tissue development and stem cell fate determination [19]. EZH2 silences the expression of multiple pathway-related genes including Wnt, thereby promoting cellular EMT, inhibiting cellular senescence and cell differen-tiation [20, 21]. EZH2 has been found to be highly expressed in various malignant tumor cells, such as breast cancer, prostate cancer, bladder cancer, lung cancer, gastric cancer, pancreatic cancer and nasopharyngeal carci-noma [22-27]. The high expression of EZH2 is

closely related to tumor growth infiltration, lymph node involvement, clinical stage and poor prognosis. Whether EZH2 has this role in LSCC is yet to be fully explored, but there is increasing evidence of the importance of EZH2 in LSCC. High EZH2 expression was shown to be associated with more malignant forms of LSCC [28].

Material and methods

Specimens

We collected specimens from all 160 patients who were diagnosed with LSCC at the First Affiliated Hospital of Bengbu Medical University, from January 2012 to January 2018. Patients underwent radical resection and peripheral mesenteric lymph node dissection. All patients were sporadic cases who had compete clinical, pathological and follow-up data (per 4 months intervals by phone, mail or out-patient follow-up) until October 2018. We excluded patients who had no complete data or history of received preoperative chemotherapy and radiotherapy. The study was approved by the ethics commit-tee of the First Affiliated Hospital of Bengbu Medical University and performed obeying the guidance of the Declaration of Helsinki. Overall survival (OS) time was calculated from patients’ surgery to death of LSCC or last follow-up (mon- ths). Tumor differentiation grade was defined according to World Health Organization. Clinical stages were rejudged based on AJCC/UICC 8th Edition new staging rules in Oropharyngeal Squamous Cell Carcinoma [29]. Clinicopatho- logic characteristics are listed in Table 1.

Immunohistochemical analysis

Immunohistochemical (IHC) staining was per-formed to explore the expression of USP7 and EZH2 in both LSCC tissues and adjacent nor-mal larynx tissues. Immunohistochemical anal-ysis was performed with the ElivisionTM Plus

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[image:3.612.90.288.87.414.2]

Table 1. Patients characteristics

Patient characteristics Frequency (n) Percentage (%) Gendel

Male 112 70.0

Female 48 30.0

Age (years)

< 60 89 55.6

≥ 60 71 44.4

Site

Glottic 96 60.0

Supraglottic 60 37.5

Subglottic 4 2.5

Tumor size

< 3 cm 91 56.9

≥ 3 cm 69 43.1

Pathologic differentiation

Well 66 41.3

Moderate 57 35.6

Poor 37 23.1

Lymphatic invasion

Negative 98 61.3

Positive 62 38.8

pTNM

I + II 75 46.9

III + IV 85 53.1

peroxidase activity, placed in citrate buffer (pH 6.0), and heated to 95°C for 30 min for antigen repair. After three washes with PBS, all sec-tions were blocked with goat serum at room temperature for 20 min, followed by incubation with rabbit monoclonal antibody against prima-ry antibodies USP7 (ab4080, Abcam, USA) and EZH2 (ab186006, Abcam, USA) at 4°C over-night. Finally, all sections were counterstained with hematoxylin, dehydrated, air-dried, and mounted.

Evaluation of staining

The staining results were interpreted by two independent pathologists who were blinded to the clinical data and were judged by semi-quan-titative points. To overcome the intra-tumoral heterogeneity of antigen expression, 10 visual fields from different areas of each tumor were examined. In cases of disagreement between the observers, consensus was achieved by re-examining the sections. Staining was scored according to the intensity and extent, < 4 as

negative expression, ≥ 4 as positive expres-sion. The staining intensity score was graded for four stages as follows: 0, none; 1, weak; 2, moderate; and 3, strong. The extent of positive staining was graded as follows: 1, ≤ 10%; 2, 11%-50%; 3, 51%-75%; and 4, > 75%.

Statistics

Chi-square test, univariate analysis, and multi-variate analysis were conducted to statistically evaluate the clinical significance of USP7 and EZH2 in LSCC. The SPSS software package (version 24.0, IBM, USA) was used for the sta-tistical analysis. The associations between protein expression and clinicopathologic para- meters were evaluated by chi-square tests. Kaplan-Meier method was used to generate survival curves, and the differences were com-pared by log-rank test. A Cox multivariate pro-portional hazard regression model was intro-duced to determine the independent effect on OS. Beta coefficients and 95% confidence intervals (CI) were used for analysis. Differences were considered significant if P < 0.05.

Results

Expression of USP7 and EZH2 in different grade of LSCC

We selected typical histochemical staining of LSCC on high-differentiation (Figure 1A and

1D), moderate differentiation (Figure 1B and

1E), and low differentiation (Figure 1C and 1F). Representative IHC result showed the negative protein expression of USP7 in normal laryngeal tissues (Figure 2A). Representative IHC result showed the high protein level of USP7 in LSCC tissues, which predominately located in the nucleus and the expression level in high-differ-entiation, moderate differhigh-differ-entiation, and low dif-ferentiation LSCC is increasing (Figure 2C, 2E

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tissue (Figure 2B). Moreover, a positive correla-tion was observed between nuclear and cyto-plasmic EZH2 staining intensity in malignant LSCC epithelial cells.

Correlations among USP7, EZH2 and clinico-pathological characteristics

Although USP7 showed a generally increased level in LSCC tissue, we classified patients into a low-USP7 group and a high-USP7 group based on the IHC scores to better investigate its clinical effects. According to the chi-square tests, high USP7 level was associated with pathologic differentiation (P < 0.001), lymphat-ic invasion (P < 0.001), and advanced TNM stage (P < 0.001) (Table 2). The correlations between high USP7 level with unfavorable cli- nicopathologic characteristics indicated that USP7 may promote tumor progression of LSCC. High EZH2 level was associated with pathologic differentiation (P < 0.05) and advanced TNM stage (P < 0.001) (Table 2). The correlations between high EZH2 level with unfavorable cli- nicopathologic characteristics indicated that EZH2 may promote tumor progression of LSCC.

Furthermore, correlation between USP7 and EZH2 showed a positive correlation with these two factors expression (r = 43.361, P < 0.001) (Table 3).

Prognostic effect of USP7 and EZH2 for LSCC patients

We plotted the survival curves using Kaplan-Meier methods based on different variables. Importantly, high USP7 level was correlated with unfavorable clinical outcomes of ESCC patients (Figure 3C). The mean overall survival time of patients in low-USP7 group was 62.37 ± 20.196 months, while only 36.24 ± 16.334 months in the patients with higher USP7 levels (Table 4, P < 0.001). High EZH2 level was cor-related with unfavorable clinical outcomes of LSCC patients (Figure 3D). The mean overall survival time of patients in low-EZH2 group was 54.27 ± 22.573 months, while it was only 37.28 ± 17.322 months in the patients with higher EZH2 levels (Table 4, P < 0.001).

[image:4.612.92.523.73.287.2]
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[image:5.612.89.373.71.501.2]

Figure 2. The presentation of immunochemical staining of normal epidemal laryneal tissue and LSCC. A: Negative staining of USP7 in laryneal epithe-lium. The cytoplasm in the granular layer is light yellow (400 magnification); B: Negative staining of EZH2 in squamous epithelium of the larynx, the nucleus in the granular layer is light yellow (400 magnification); C: Negative staining of USP7 in hyperplastic squamous cells. There is flaky expression in the cytoplasm, as well as staining of individual nuclei (400 magnifica-tion); D: Negative staining of EZH2 in hyperplastic squamous cell, mainly expressed in the nucleus and cytoplasm, but the degree of staining is light (400 magnification); E: Positive staining of USP7 in moderate differentia-tion, diffusely expressed in the cytoplasm and nucleus with brown-staining (400 magnification); F: Positive staining of EZH2 in moderate differentia-tion, diffusely expressed in the nucleus with brown staining (400 magnifica-tion); G: Positive staining of USP7 in poorly differentiation, full expression in the cytoplasm and nucleus, dark brown (400 magnification); H: Positive staining of EZH2 in poor differentiation, full expression in nucleus, dark-brown staining (400 magnification).

LSCC. The pTNM showed no significantly inde-pendent effect (Table 5, P = 0.234). However, lymphatic invasion was independently

correlat-Ubiquitination modifications can regulate al- most all physiologic or pathologic processes. USP7/HAUSP, is a deubiquitinating enzyme that

ed with a poor overall survival (HR = 2.605, 95% CI 1.534-4.423, P < 0.001). Furthermore, low USP7 expression was an independent protective factor for the clinical outcome (HR = 4.338, 95% CI 1.639-11.477, P = 0.003). Low EZH2 expres-sion was also an independent protective factor for the clinical outcomes (HR = 2.130, 95% CI 1.063-4.269, P = 0.033) (Table 5).

Discussion

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regulates many key proteins such as tumor sup-pressor genes, DNA repair proteins, immune responders, viral proteins, and epigenetic regu-lators [6, 9, 35]. The role of biological activity, USP7 abnormalities can cause tumor and viral diseases, therefore, it has become a potential therapeutic target [36]. Ubiquitin specific prote-ases (USP) are among the most studied mem-bers of the DUBs family, including more than 60 members. Current studies have found that several USP family members are closely related to tumorigenesis and metastasis, such as USP1 and glioblastoma (GBM) [37]; USP2, USP11 and breast cancer [38, 39], USP7 with prostate cancer [40]; and USP4 with colorectal cancer [41].

In view of the important role of EZH2 protein in tumorigenesis, it can be used as a treatment for tumors. A potential rough point provides a new research direction for the precise treat-ment of tumor diseases. However, the exact mechanism that plays a role in the growth and metastasis of EZH2 protein tumors remains the focus of future research. Expression array stud-ies in lymphoma cells treated with EZH2 inhibi-tors have shown mostly increases in gene expression [42, 43], as would be expected given its silencing role. What is the relationship between EZH2-mediated histone methylation and DNA methylation and will targeting one be sufficient to overcome tumor suppressor gene silencing? Further understanding of this pro-cess may help guide combination treatments with EZH2 inhibitors.

In our study high USP7 level was correlated with unfavorable clinical outcomes of ESCC patients and high EZH2 level was also correlat-ed with unfavorable clinical outcomes of LSCC patients. Both USP7 and EZH2 level were

asso-Table 2. Relationship between expression of USP7 and EZH2 and clinicopathologic characteristics of laryngeal squamous cell carcinoma

Characteristic USP7 P value EZH2 P value

Negative Positive Negative Positive

Gender

Male 27 85 0.297 33 79 0.395

Female 8 40 11 37

Age (years)

< 60 24 65 0.081 31 58 0.020

≥ 60 11 60 13 58

Site

Glottic 17 79 0.163 28 68 0.846

Supraglottic 16 44 15 45

Subglottic 2 2 1 3

Tumor size

< 3 cm 24 67 0.114 30 61 0.075

≥ 3 cm 11 58 14 55

Pathologic differentiation

Well 25 41 < 0.001 24 42 0.020

Moderate 7 50 16 31

Poor 3 34 4 33

Lymphatic invasion

Negative 31 67 < 0.001 32 66 0.066

Positive 4 58 12 50

pTNM

I + II 30 45 < 0.001 31 44 < 0.001

[image:6.612.92.523.96.426.2]

III + IV 5 80 13 72

Table 3. Correlation between USP7 and EZH2

Variable USP7 r P

Negative Positive EZH2

[image:6.612.90.288.462.530.2]
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ciated with pathologic differentiation (P < 0.001). Furthermore, correlation between USP7 and EZH2 showed a positive correlation with these two factors expression. USP7 and EZH2 may promote tumor progression of LSCC. Studies have reported that USP7 has many substrates involved in the development of can-cer, the most common of which is the USP7-MDM2-P53 molecular axis [44]. Another

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[image:8.612.92.349.94.504.2]

Table 4. Results of univariate analyses of overall survival (OS) time

Variable n Mean OS (months) rankLog- P-value USP7

Negative 35 62.37 ± 20.196 27.772 < 0.001 Positive 125 36.24 ± 16.334

EZH2

Negative 44 54.27 ± 22.573 17.338 < 0.001 Positive 116 37.28 ± 17.322

Gender

Male 112 41.96 ± 20.193 0.081 0.776

Female 48 41.94 ± 20.816

Age (years)

< 60 89 43.30 ± 21.007 0.295 0.587

≥ 60 71 40.27 ± 19.430

Site

Glottic 96 38.93 ± 19.362 1.649 0.438 Supraglottic 60 45.98 ± 21.144

Subglottic 4 54.25 ± 19.805 Tumor size

< 3 cm 91 43.12 ± 20.971 0.074 0.786

≥ 3 cm 69 40.42 ± 19.463

Pathologic differentiation

Well 66 46.23 ± 23.076 5.036 0.081

Moderate 57 41.42 ± 17.675

Poor 37 35.16 ± 17.150

Lymphatic invasion

Negative 98 43.57 ± 21.888 30.432 < 0.001 Positive 62 39.40 ± 17.413

pTNM

I + II 75 43.20 ± 23.785 8.256 0.004

III + IV 85 40.86 ± 16.741

bilize its protein expression, but further study found that USP7 preferentially forms a stable complex with MDM2 in cells, which promotes the degradation of P53 protein [48]. The high expression of USP7 in tumor cells is mainly due to the increased stability of MDM2 and pro-motes tumor development. The regulation of p53 by USP7 is very complex. On the one hand, USP7 deubiquitinates p53, which prevents p53 from being deubiquitinated and is degraded by the proteasome, thereby increasing the intra-cellular level of p53. On the other hand, USP7 can also deubiquitinate the negative regulatory proteins of p53, such as MDM2 and MDM4, and increase the intracellular level of p53 negative regulatory protein, which leads to a

of small molecule inhibitors have been identi-fied through a proprietary screening platform, which has the characteristics of stabilizing p53 expression, inhibiting cancer cell proliferation and activity in disease cell models [13, 53]. Whether USP7 and EZH2 promote the invasion and metastasis of LSCC through the interaction mechanism of p53, we still need a more com-plete experimental system to verify, and the specific mechanism is waiting for us to discover.

Conclusions

Our findings imply that USP7and EZH2 affects LSCC evolution; USP7 and EZH2 are

upregulat-decrease in the intracellular level of p53 and a decrease in activity. EZH2 can play a role in tumor inva-sion and metastasis by affecting cytoskeletal polymerization inde-pendent of its histone methyltrans-ferase activity [49]. P53 alteration may be involved in dysregulated EZH2 expression, and aberrant ex- pression of EZH2 may play a role in carcinogenesis of oral squamous cell carcinoma [50]. P53 can inhibit the promoter of EZH2 and inhibit the overexpression of EZH2. On the other hand, low expression of EZH2 can effectively prolong the G2/M phase of the cell cycle and impede cell proliferation [51].

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ed in LSCC tissues, which can serve as an inde-pendent prognostic predictor. USP7 and EZH2 are both target biomarkers of LSCC and provide the possibility for developing inhibitors to treat LSCC.

Acknowledgements

This work was supported by the Nature Sci- ence Foundation of China (No. 81072207) and Anhui Provincial University Natural Science Key Project (No. KJ2015A284).

Disclosure of conflict of interest

None.

Address correspondence to: Yue-Feng Han, Depart- ment of Otorhinolaryngology, The First Affiliated Hospital of Bengbu Medical University, No. 287, Changhuai Road, Anhui Province, China. Tel: +86-13500567009; Fax: +86-05523086359; E-mail: HYFBBYXY@163.com

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Figure

Table 1. Patients characteristics
Figure 1. Presentation of histochemical staining of high-differentiation, moderate differentiation, and low differen-tiation
Figure 2. The presentation of immunochemical staining of normal epidemal laryneal tissue and LSCC
Table 2. Relationship between expression of USP7 and EZH2 and clinicopathologic characteristics of laryngeal squamous cell carcinoma
+4

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